MIR27A Gene Polymorphism Modifies the Effect of Common DPYD Gene Variants on Severe Toxicity in Patients with Gastrointestinal Tumors Treated with Fluoropyrimidine-Based Anticancer Therapy

To reduce severe fluoropyrimidine-related toxicity, pharmacogenetic guidelines recommend a dose reduction for carriers of four high-risk variants in the DPYD gene (*2A, *13, c.2846A>T, HapB3). The polymorphism in the MIR27A gene has been shown to enhance the predictive value of these variants. Our study aimed to explore whether rs895819 in the MIR27A gene modifies the effect of five common DPYD variants: c.1129-5923C>G (rs75017182, HapB3), c.2194G>A (rs1801160, *6), c.1601G>A (rs1801158, *4), c.496A>G (rs2297595), and c.85T>C (rs1801265, *9A). The study included 370 Caucasian patients with gastrointestinal tumors who received fluoropyrimidine-containing chemotherapy. Genotyping was performed using high-resolution melting analysis. The DPYD*6 allele was associated with overall severe toxicity and neutropenia with an increased risk particularly pronounced in patients carrying the MIR27A variant. All carriers of DPYD*6 exhibited an association with asthenia regardless of their MIR27A status. The increased risk of neutropenia in patients with c.496G was only evident in those co-carrying the MIR27A variant. DPYD*4 was also significantly linked to neutropenia risk in co-carriers of the MIR27A variant. Thus, we have demonstrated the predictive value of the *6, *4, and c.496G alleles of the DPYD gene, considering the modifying effect of the MIR27A polymorphism.


Introduction
Fluoropyrimidines (FPs), including 5-fluorouracil and capecitabine, are widely used in the treatment of various solid tumors as part of chemotherapy regimens.Despite their proven efficiency, the use of FPs commonly leads to severe toxicity, followed by interruptions and delays in chemotherapy delivery, life-threatening events and even death [1].
A well-known cause of severe FP-related toxicity is the deficiency of dihydropyrimidine dehydrogenase (DPD) activity as far as DPD plays a crucial role in metabolizing these drugs.Numerous studies have identified four allelic variants in the DPYD gene that encode DPD, leading to decreased or absent DPD enzymatic activity, and they are associated with severe FP toxicity with a high level of evidence: *2A (rs3918290), *13 (rs55886062), -2846A>T (rs67376798), and c.1129-5923C>G/HapB3 (rs75017182) [2,3].Pharmacogenetic guidelines recommend a 25-50% dose reduction or avoidance of FPs for carriers of these variants [2][3][4].Several prospective studies demonstrated that a genotype-based treatment approach (namely, drug dose reduction in DPD-deficient patients) could drastically reduce the incidence of severe toxicity in carriers without compromising on chemotherapy efficacy [5][6][7][8].However, pre-emptive genotyping is not always beneficial [9].
An obvious limitation of the current genotype-based approach is that the four recommended DPYD variants are relatively rare and may explain only a small part of severe FP-related toxicity.Consequently, there is ongoing research into other DPYD gene variants that could improve toxicity risk prediction [10][11][12].While some studies have thoroughly investigated rare variants of the DPYD gene [13], the clinical applicability of this new knowledge remains uncertain.On the other hand, more common variants of the DPYD gene appear to be promising markers for improving the prediction of FP-related toxicity.For example, alleles *4 (c.1601G>A, rs1801158), *6 (c.2194G>A, rs1801160), *9A (c.85T>C, rs1801265), and c.496A>G (rs2297595) of the DPYD gene have been associated with an increased risk of FP-related toxicity in several studies [10,[14][15][16][17].However, the impact and clinical validity of these and other alleles on FP-related toxicity requires further investigation, as evidence is currently insufficient or contradictory [18].
A promising approach to enhance the predictive value of DPYD variants is analyzing the polymorphism of the MIR27A gene, which encodes the microRNA miR-27a.This microRNA regulates DPD activity at the post-transcriptional level by inhibiting the expression of DPYD mRNA [19][20][21].The variant allele rs895819 (A>G) in the MIR27A gene is associated with an increased expression of the MIR27A gene [19].This variant has been shown to significantly improve the accuracy of toxicity prediction in carriers of the four recommended DPYD gene variants [22].The combination of a high-risk DPYD gene variant and the minor G allele of rs895819 in the MIR27A gene significantly increased the risk of FP-related toxicity [22,23].Moreover, patients with DPYD risk variants who received appropriate genotype-specific dose reductions remained at increased risk of fluoropyrimidine toxicity if they carried rs895819 in the MIR27A gene [21].However, the effect of the MIR27A gene polymorphism on FP-related toxicity has primarily been studied in conjunction with the carriage of the four recommended DPYD variants [21][22][23].We hypothesized that similar analyses might be useful for understanding the functional role and potential clinical utility of other common DPYD variants.Our research aimed to investigate whether the rs895819 variant in the MIR27A gene modifies the effect of five DPYD variants (c.1129-5923C>G, c.2194G>A, c.1601G>A, c.496A>G, c.85T>C) on the risk of FP-related toxicity.

Characteristics of Studied Population
The prospective observational study included 370 patients with gastrointestinal tumors who received chemotherapy containing FPs (Table 1).Of these, 55 patients (14.9%) were treated with capecitabine-based regimens.
This study collected data on the incidence of overall severe toxicity (grade ≥ 3), neutropenia (grade ≥ 3), and asthenia (grade ≥ 2), which were each analyzed separately.Toxicity was assessed at two endpoints: (1) after the first course (early toxicity) and (2) after 4 courses, including all toxicities during courses 1-4.The choice of courses 1 to 4 as a secondary endpoint was based on the assumption that the majority of patients have first follow-up examination after 4 courses of therapy.Thus, the majority of patients (n = 333) received at least four courses of FP-containing chemotherapy.This selection of endpoints allows for the assessment of early toxicity as well as the cumulative impact of chemotherapy on the development of toxicity while reducing attrition bias or the influence of varying chemotherapy courses among patients.Thirty-seven patients received fewer than four courses of FPs due to disease progression, a change in therapy, or intolerable toxicity.For these patients, when assessing toxicity in courses 1-4, cases of toxicity were recorded if they occurred while receiving FPs.Otherwise, the data were considered not available (NA).The development of toxicity was as follows: after the first course of FP-containing therapy, overall severe toxicity was observed in 62 patients (16.8%), including neutropenia in 27 (7.3%)and asthenia in 52 (14%).During courses 1-4, overall severe toxicity was observed in 148 of 352 patients (42%), neutropenia in 73 of 328 (22.3%), and asthenia in 83 of 328 (25.3%).

Genotypes of Studied Population
The genotypes were successfully determined in 367 patients (Table 2).The genotypes of three samples could not be determined due to insufficient DNA quality.The genotyping results for all markers were found to be in accordance with the Hardy-Weinberg equilibrium.

Identification of Clinical and Anthropometric Factors Influencing Toxicity
For each type of toxicity, we identified which clinical and anthropometric factors influenced its occurrence to further use significant (p-value < 0.05) variables as covariates in analyzing the influence of genetic variants on the risk of toxicity.The effects of sex, age, weight, body mass index (BMI), comorbidities, Eastern Cooperative Oncology Group (ECOG) performance status, and concomitant use of taxanes, oxaliplatin, irinotecan, and targeted therapy were assessed in a univariate analysis using logistic regression.
Regarding the first course toxicity endpoint, the co-administration of taxanes was a significant predictor of overall severe toxicity; significant predictors of neutropenia were the comorbidity and co-administration of taxanes; significant predictors of asthenia included sex (female), ECOG status, and weight (Table S1).Regarding adverse events after four courses of FP-containing chemotherapy, significant predictors of overall severe toxicity included sex (female), ECOG status, and the co-administration of irinotecan or taxanes; for neutropenia, significant factors were weight and co-administration of irinotecan or taxanes; for asthenia, predictors included sex (female), ECOG status, weight, age, and comorbidity (Table S1).Thus, these variables were used as covariates in multivariate logistic regression analysis to assess the role of genetic variants in the occurrence of these toxicities.

The Association of Genetic Markers with FP-Related Toxicity
We analyzed the impact of the studied genetic variants on FP-related toxicity using a dominant model, where carriers of variant alleles were compared with wild-type (wt) homozygotes.The modifying effect of the MIR27A gene polymorphism on the risk of toxicity in carriers of each studied DPYD gene variant was also analyzed.Carriers of DPYD gene variants were categorized into those who had the variant allele (G) of the MIR27A gene (MIR27A + ) and those who did not (MIR27A − ), and comparisons were made with wt-homozygotes.If the statistical significance of the DPYD allele varied depending on the presence of the MIR27A variant, interaction analysis was conducted.
All analyses were performed using multivariate logistic regression, incorporating significant clinical and anthropometric predictors (Table S1) as covariates.Consequently, the resulting odds ratios (ORs) and p-values were adjusted for risk factors associated with the corresponding types of toxicity.

The Association of Genetic Markers with Early FP-Related Toxicity
The results of the analysis of genetic markers with early FP-related toxicity are presented in Table 3.
Other genetic markers included in the study showed no association with early FPrelated toxicity.

The Association of Genetic Markers with FP-Related Toxicity after 4 Courses
The analysis of genetic factors during the first four courses of FP-containing chemotherapy is summarized in Table 4.
No other genetic markers were associated with FP-related toxicity during the first four courses.

Discussion
Current guidelines for pharmacogenetic testing recommend the identification of four allelic variants of the DPYD gene (*2A-rs3918290, *13-rs55886062, -2846A>T-rs67376798, and -1236G>A/HapB3-rs56038477) before prescribing FPs [2][3][4].For carriers, a dosage reduction or avoidance of these drugs is recommended.However, it is clear that these variants, due to their low frequency, cannot explain all cases of severe toxicity, even those mediated by a genetically determined decrease in DPD activity.
We found it encouraging that rs895819 in the MIR27A gene significantly improves the predictive value of high-risk DPYD variants for identifying patients at risk of severe FP-related toxicity [21][22][23].Although these studies mainly included the four recommended DPYD variants, the article by Meulendijks et al. [23] also included the DPYD*4 allele.We hypothesized that a similar approach could enhance the significance and reproducibility for other variants in the DPYD gene that affect DPD activity.In the present study, we examined the association of five common variants in the DPYD gene with severe FP-related toxicity in patients with gastrointestinal tumors.Assuming that the effect of these genetic markers may depend on the status of the MIR27A gene, we also analyzed the studied alleles depending on the status of the MIR27A gene: without or together with the variant MIR27A allele (MIR27A − and MIR27A + , respectively).
The most pronounced role as a genetic risk factor for severe FP-related toxicity in our study was demonstrated by the DPYD*6 allele.This allele has been associated with various types of FP-related toxicity in numerous studies [10,[14][15][16][24][25][26][27].Although some researchers have reported negative results [28][29][30][31][32], a relatively recent meta-analysis confirmed the significance of this allele in European patients [33].Our data suggest that the risk effect of DPYD*6 on FP-related toxicity may be modified by the presence of the MIR27A rs895819 variant, although this interaction varies across different types of toxicity.We observed an association of DPYD*6 with neutropenia at both endpoints (at course 1 and during courses 1-4), and the overall severe toxicity during courses 1-4 was found, specifically in patients carrying the MIR27A variant, but not in those without it.Direct confirmation of the interaction between these variants was obtained for the risk of neutropenia in the first four courses of FP-containing chemotherapy.On the other hand, the risk of asthenia (grade ≥ 2) in courses 1-4 was increased in all carriers of the DPYD*6 allele regardless of their MIR27A status.Further studies in larger cohorts are necessary to clarify the clinical relevance of the interaction between DPYD*6 and MIR27A.We suppose that DPYD*6 is indeed a promising marker for predicting FP-related toxicity and that testing for MIR27A could enhance its predictive value.
Another variant that showed an association with toxicity in our study was c.496G.The previously published results were controversial: while some studies described c.496G as a risk factor [16,17,27,30,34], others reported its protective effect [35], and several presented negative results [10,15,28,32,36,37].We found that the risk of neutropenia in carriers of this allele is statistically significantly associated with the co-presence of the MIR27A variant.In patients with both 496G and MIR27A + alleles, the risk of neutropenia in the first cycle was increased, whereas for c.496G and MIR27A − patients, no association was noted, as well as in the overall group of c.496G carriers.Therefore, the interaction with MIR27A status may provide a more accurate assessment of the association of the c.496G variant with FP-related toxicity.
Additionally, neutropenia in courses 1-4 was associated with the DPYD*4 allele, and its effect was evident across the entire carrier group, as well as for patients with *4 and MIR27A + , but not for those with *4 and MIR27A − .However, in our study, the interaction of these variants was not significant, which might be explained by the insufficient sample size.Interestingly, a meta-analysis by Meulendijks et al. [23] showed that in patients with DPYD*4 (c.1601G>A), MIR27A variants were associated with the risk of severe FPassociated toxicity, and our results are consistent with these observations.It is worth noting that despite contradictory data, DPYD*4 is already used in preemptive testing in some studies [38], although data on its role are contradictory [39].
Other genetic markers included in our study, such as HapB3 and *9A in the DPYD gene, showed no association with severe FP-related toxicity.We examined the HapB3 variant, which is included in pharmacogenetic guidelines because they recommend an FP dose reduction only for homozygotes, which is extremely rare [2,3].In a meta-analysis by Meulendijks et al., carriers of HapB3 were shown to have an increased risk of severe toxicity when also carrying the MIR27A variant [23].However, we did not find an association of HapB3 with severe toxicity even when considering the MIR27A status.
The association of the DPYD*9A allele with FP-related toxicity is quite controversial according to published data: there is evidence of a risk [17,29], a protective effect [10,40], and no effect [16,27,28,30,41].The analysis of this variant is complicated by the fact that it is linked to other SNPs, particularly c.496A>G, and there is evidence of the influence of the haplotype on enzyme activity and, consequently, the risk of toxicity [40,42,43].We did not perform haplotype analysis in our study.
In our study, rs895819 in the MIR27A gene alone had no effect on the risk of FPrelated toxicity but did show a modifying effect for some DPYD variants.We included only rs895819 in the MIR27A gene in our study, while a number of other studies also included rs11671784.However, rs11671784 was found to be less significant, occurs at a lower frequency [21][22][23], and is in strong linkage disequilibrium with rs895819 [23].Therefore, we decided not to include it in our study.
The role of MIR27A variants has differed between studies.Amstutz et al. [22] reported a protective effect in patients without DPYD risk variants and an increased risk in carriers, but a subsequent meta-analysis by Meulendijks et al. showed an association with risk in carriers and in the overall population but no association in DPYD wild-type patients [23].According to Falvella et al., severe side effects of FPs were associated with homozygous rs895819 [34], whereas Medwid et al. and Ragia et al. showed an increased risk in heterozygotes [21,44].
Thus, the data already published and obtained in the present study indicate that the variant allele rs895819 may have a risk effect that is more pronounced in carriers of dysfunctional DPYD variants.These results are logically consistent with the mechanisms underlying the influence of miR-27a on DPD activity.It is known that miR-27a targets the 3 ′ -untranslated region of DPYD mRNA (this gene is one of its targets), inhibiting its translation [19][20][21]45].MiR-27a expression has been found to be inversely correlated with DPYD mRNA levels and DPD activity [19].The variant G allele of rs895819 leads to an increased expression of the MIR27A gene and, consequently, a decrease in DPD enzyme activity [19].
This study has some limitations.We selected DPYD gene variants based on a narrative review of the literature, but the testing of an expanded range of genetic markers seems to be reasonable in the future larger studies.No preliminary calculation of sample sizes was carried out, which was primarily because of the varying frequencies of minor alleles of the studied markers and the unknown effect size for most of them.When planning further validation studies, it is necessary to estimate sample sizes in advance to ensure the required statistical power.This study included only Caucasians, but further research is needed to confirm these findings in larger and more diverse populations.

Patients
This prospective observational study included Caucasian patients with gastrointestinal tumors (gastric cancer, colorectal cancer, pancreatic cancer, small intestine cancer, esophageal cancer, and liver cancer), who received FP-based chemotherapy at the City Clinical Hospital named after S.S. Yudin of the Moscow Health Department (Chemotherapy Department No. 1) from October 2020 to May 2022.Exclusion criteria were age younger than 18 years and a history of previous anticancer chemotherapy or radiotherapy.All the patients signed an informed consent to participate in the study.The study was conducted in accordance with the Declaration of Helsinki and approved by the Local Ethics Committee of the Russian Medical Academy of Continuous Professional Education of the Ministry of Healthcare, Moscow, Russian Federation (protocol No. 9, date of approval 7 July 2020).The collection of blood samples and prospective follow-up of the patients was carried out under the Russian Science Foundation project No. 20-75-10158.All the patients underwent pharmacogenetic testing of the DPYD*2A allele before the 1st course of chemotherapy.Carriers of the DPYD*2A allele were not included in this study.
Toxicity was assessed prospectively according to the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) v5.0.

Genotyping
DNA was isolated from venous blood samples collected in EDTA-containing tubes using QIamp DNA Mini kits (Qiagen, Hilden, Germany).The concentration of DNA was assessed using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

Statistical Analysis
Statistical analysis was performed using R version 4.3.1 (R Foundation for Statistical Computing, Vienna, Austria).The influence of clinical and anthropometric factors on toxicity was assessed using univariate logistic regression.The effect of genetic variants on toxicity was tested using multivariate logistic regression with statistically significant clinical and anthropometric factors used as covariates.An exact test for Hardy-Weinberg equilibrium was performed using the "HardyWeinberg" package (version 1.7.8).Differences were considered statistically significant when the p-value was below 0.05.No adjustment for multiple comparisons was applied.

Conclusions
Our findings suggest that the risk of FP-related toxicity in DPYD *4, *6, and c.496G carriers may depend on the rs895819 MIR27A variant.Thus, we demonstrated that an approach accounting for the modifying effect of the MIR27A gene polymorphism is promising when studying the association of DPYD variants with FP-related toxicity.We believe that this strategy may be useful in studying other genetic markers in the DPYD gene in future research, ultimately improving the accuracy of existing approaches to predicting toxicity based on the patient's genotype and enhancing the safety of FP-containing chemotherapy.
Supplementary Materials: The supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ijms25158503/s1.Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Author Contributions:
Conceptualization, A.I., M.E., D.F., and V.L.; methodology, A.I., A.M., R.H., and D.F.; software, R.H.; validation, M.L., and V.M.; formal analysis, V.M.; investigation, A.I., and D.F.; resources, V.M., and V.L.; data curation, A.M., and M.L.; writing-original draft preparation, A.I., M.E., and D.F.; writing-review and editing, D.G., A.M., and V.L.; supervision, M.E., and D.G.; project administration, V.L.; funding acquisition, D.G.All authors have read and agreed to the published version of the manuscript.Funding: This work was supported by the Ministry of Science and Higher Education of the Russian Federation to the EIMB Center for Precision Genome Editing and Genetic Technologies for Biomedicine under the Federal Research Program for Genetic Technologies Development for 2019-2027 [Agreement No. 075-15-2019-1660].Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki and approved by the Local Ethics Committee of the Russian Medical Academy of Continuous Professional Education of the Ministry of Healthcare, Moscow, Russian Federation (protocol No. 9, date of approval 7 July 2020).

Table 1 .
Baseline characteristics of patients with gastrointestinal tumors.

Table 2 .
DPYD and MIR27A genotyping in patients with gastrointestinal tumors.

Table 3 .
Risk of early fluoropyrimidine-associated toxicity according to DPYD and MIR27A status.